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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Echicetin, a GPIb-binding snake C-type lectin from Echis carinatus, also contains
a binding site for IgM␬ responsible for platelet agglutination
in plasma and inducing signal transduction
Alexei Navdaev, Dagmar Dörmann, Jeannine M. Clemetson, and Kenneth J. Clemetson
Echicetin, a heterodimeric snake C-type
lectin from Echis carinatus, is known to
bind specifically to platelet glycoprotein
(GP)Ib. We now show that, in addition, it
agglutinates platelets in plasma and induces platelet signal transduction. The
agglutination is caused by binding to a
specific protein in plasma. The protein
was isolated from plasma and shown to
cause platelet agglutination when added
to washed platelets in the presence of
echicetin. It was identified as immunoglogulin M␬ (IgM␬) by peptide sequenc-
ing and dot blotting with specific heavy
and light chain anti-immunoglobulin reagents. Platelet agglutination by clustering echicetin with IgM␬ induced P-selectin expression and activation of GPIIb/IIIa
as well as tyrosine phosphorylation of
several signal transduction molecules,
including p53/56LYN, p64, p72SYK, p70 to
p90, and p120. However, neither ethylenediaminetetraacetic acid nor specific
inhibition of GPIIb/IIIa affected platelet
agglutination or activation by echicetin.
Platelet agglutination and induction of
signal transduction could also be produced by cross-linking biotinylated
echicetin with avidin. These data indicate
that clustering of GPIb alone is sufficient
to activate platelets. In vivo, echicetin
probably activates platelets rather than
inhibits platelet activation, as previously
proposed, accounting for the observed
induction of thrombocytopenia. (Blood.
2001;97:2333-2341)
© 2001 by The American Society of Hematology
Introduction
Snakes produce venoms containing a wide variety of components
that kill or weaken their prey. Whereas venoms from some snake
families contain mostly neurotoxic proteins, others such as the
Viperidae and Crotalidae genera are mainly hemorrhagic. Among
the protein families that have been shown to have hemorrhagic
effects are the snake C-type (calcium-dependent) lectins. This
family is named after the type of folding that occurs in classic
C-type lectins such as mannose-binding protein1,2 and the selectins.3 Many snake C-type lectins have now been characterized with
effects on either coagulation factors or platelets. Those affecting
platelets either inhibit or activate them by binding to specific
receptors like glycoprotein (GP)Ib, ␣2␤1, and GPVI. Those that act
via GPIb to agglutinate platelets include alboaggregins,4-6 flavocetin-A and -B,7 and mamushigin.8 Most of the inhibitory C-type
lectins described so far bind to GPIb. These include echicetin,9
jararaca GPIb-binding protein,10,11 tokaracetin,12 CHH-A and -B,4
and agkicetin.13 Echicetin, a heterodimeric snake C-type lectin
from Echis carinatus, has been shown by several authors to bind
specifically to platelet GPIb and to block platelet interactions with
von Willebrand factor (vWf)9 and with thrombin.14 There has been
considerable interest in using C-type lectins, such as echicetin as
antithrombotics, in blocking the interaction between vWf and
platelets. However, when echicetin or similar snake C-type lectins
have been injected into small animals to study their effects in vivo,
induction of thrombocytopenia has often been reported.9,15 Generally, the platelet count dropped to 20% to 30% of the control value
and then gradually recovered over several hours. This phenomenon
has remained unexplained. In addition, it is far from clear why a
snake venom component blocking GPIb as single mode of action
would have evolved. GPIb is one of the most common platelet
receptors, with at least 25 000 copies per platelet (and more likely
50 000 based on monomeric snake C-type lectin binding), and
needs to be inhibited to at least 80% to effect platelet function. The
number of snake venom component molecules required to inhibit
80% of GPIb on all platelets in the circulation of even a small
animal is quite considerable and would be an inefficient strategy for
producing bleeding in the prey. Therefore, it seemed much more
likely that this category of C-type lectins causes platelet activation
by additional effects. In this paper we report that echicetin induces
platelet agglutination in platelet-rich plasma (PRP) via a multimeric, plasma protein present in microgram amounts per milliliter.
This protein was isolated and characterized, and its effects together
with echicetin on platelets were investigated in detail.
Materials and methods
Materials
Lyophilized Echis carinatus sochureki and Trimeresurus albolabris venoms
were from Latoxan (Rosans, France), protein A–Sepharose, bovine serum
albumin, ristocetin, peroxidase-conjugated rabbit antimouse antibodies,
biotinamidocaproate N-hydroxysuccinimide ester, bovine thrombin, and
fluorescein isothiocyanate (FITC) were from Sigma (Buchs, Switzerland).
N-ethylmaleimide and N-acetylglucosamine were from Fluka (Buchs,
Switzerland). Octanyl N-methylglucamide was from Oxyl Chemie (Bobingen, Germany). Human fibrinogen (vWf and plasminogen-free) was from
From the Theodor Kocher Institute, University of Berne, Switzerland.
Freiestrasse 1, CH-3012 Berne, Switzerland; e-mail: [email protected].
Submitted September 20, 2000; accepted December 14, 2000.
Supported by Swiss National Science Foundation grant 31-52396.97.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Reprints: K. J. Clemetson, Theodor Kocher Institute, University of Berne,
© 2001 by The American Society of Hematology
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
2333
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2334
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
NAVDAEV et al
Enzyme Research Labs (South Bend, IN). Fibrinogen was conjugated to
FITC as described earlier.16 Avidin was from Imtec (Moscow, Russia).
FITC-coupled chicken anti–P-selectin (CD62P) and FITC-coupled chicken
immunoglobulin Y (IgY) as control were from WAK-Chemie Medical (Bad
Soden, Germany). The peptide GPRP (Gly-Pro-Arg-Pro) was from Calbiochem-Novabiochem (Bad Soden, Germany). Sephadex G-10 and Sepharose
4B were from Pharmacia Fine Chemicals (Uppsala, Sweden). Autoradiography films were from Fujifilm (Dielsdorf, Switzerland). Antiphosphotyrosine monoclonal antibody (4G10) and anti–phosphatidylinositol-3 kinase
(PI-3K; 85-kd subunit) monoclonal antibody were from Upstate Biotechnology (Lake Placid, NY). Anti-p72SYK (4D10) monoclonal antibody, antipp125FAK (A-17) polyclonal antibodies, and anti-p53/56LYN rabbit polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
The anti-GPIb␣ monoclonal antibody (SZ2) was from Coulter-Immunotech
Diagnostics (Hamburg, Germany), and the monoclonal antibody to the
thrombin-binding site on GPIb␣, VM16d, was a kind gift from Dr A.V.
Mazurov. FITC-labeled anti-CD36 monoclonal antibody (clone FA6.152)
was from Immunotech (Marseille, France). The GPIIb-IIIa inhibitor
Ro44-9883 and the anti-GPIb␣ monoclonal antibody, Ib-4, were kind gifts
from Dr Beat Steiner, Hoffmann-La Roche (Basel, Switzerland). The
adenosine 5⬘-diphosphate (ADP) receptor inhibitor AR-C66096 was a kind
gift from Dr Bob Humphries, AstraZeneca (Loughborough, England).
Polyvinylidene fluoride (PVDF) membranes were PolyScreen from DuPont
NEN (Zaventem, Belgium). Alboaggregin A was purified from Trimeresurus albolabris venom by a method similar to that of Peng et al.17
Purification of echicetin
Lyophilized Echis carinatus sochureki venom was dissolved at 50 mg/5 mL
in 50 mM sodium acetate, pH 5.0 (buffer A). Insoluble components were
removed by centrifugation, and supernatant was loaded on a Fractogel
EMD SO3-650(S) column (10 ⫻ 150 mm, Merck, Darmstadt, Germany)
equilibrated with buffer A. Elution of echicetin was performed by a 0 to 1 M
gradient of NaCl in buffer A. Fractions (5 mL) were collected at 1 mL/min
flow rate. Activity of echicetin was determined by its ability to block
alboaggregin A–induced agglutination of fixed platelets. The fractions
containing echicetin were pooled and concentrated by SpeedVac. Further
purification of the fractions containing echicetin was performed using
reverse-phase chromatography (wide pore C-4).
Biotinylation of echicetin
Purified echicetin was dialyzed against 10 mM Na phosphate buffer, pH 8.0.
Biotinamidocaproate N-hydroxysuccinimide ester in dimethyl sulfoxide (2
mg/mL) was added to echicetin at a molar ratio 2:1. The mixture was
incubated at room temperature for 2 hours. Biotin-echicetin conjugate was
separated from free biotin by gel filtration on a Sephadex G-10 column.
Protein determination
Protein determination was performed by the bovine serum albumin protein
assay (Pierce, Sochochim, Lausanne, Switzerland) with bovine serum
albumin as standard.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
and silver staining
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
was performed by the method of Laemmli,18 and the gels were silverstained by the method of Morrissey.19
Preparation of washed platelets and platelet aggregation
Human platelets were isolated from buffy coats less than 20 hours after
blood collection obtained from the Central Laboratory of the Swiss Red
Cross Blood Transfusion Service. To one buffy coat was added 30 mL of
100 mM citrate, pH 6.5. PRP and the platelet pellet were isolated by
successive centrifugation steps. Platelets were resuspended in 113 mM
NaCl, 4.3 mM K2HPO4, 4.3 mM Na2HPO4, 24.4 mM NaH2PO4, and 5.5
mM glucose (pH 6.5) (buffer B) and centrifuged at 250g for 5 minutes. The
platelet-rich supernatant was centrifuged at 1000g for 10 minutes, and
platelets were washed with buffer B once more. Washed platelets were
resuspended in 20 mM HEPES, 140 mM NaCl, 4 mM KCl, and 5.5 mM
glucose (pH 7.4) (buffer C), and the platelet count was adjusted to
5 ⫻ 108/mL by dilution with buffer C. Samples were kept at room
temperature until used for aggregation studies. Platelet aggregation was
monitored by light transmission in an aggregometer (Lumitec, France) with
continuous stirring at 1100 rpm at 37°C. Platelets were preincubated in
buffer C containing 2 mM CaCl2 and 2 mM MgCl2 at 37°C for 2 minutes
before starting the measurement by adding the samples for analysis. All
experiments were repeated at least 3 times with platelets from different donors.
Platelet biotinylation, Triton X-100 platelet lysate, wheat germ
agglutinin affinity chromatography, and echicetin
affinity chromatography
Human platelets were isolated from buffy coats as described above but in
the presence of 10 ␮M Iloprost. Washed platelets were diluted with
phosphate-buffered saline to 5 ⫻ 109/mL and incubated with 10 ␮g
biotinamidocaproate N-hydroxysuccinimide ester for 1 hour at room
temperature. Free biotinamidocaproate N-hydroxysuccinimide ester was
removed by washing the platelets 3 times with phosphate-buffered saline,
pH 6.8. Biotinylated platelets were solubilized in phosphate-buffered saline
containing 1.2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 100
␮M leupeptin, 2 mM N-ethylmaleimide, and 2 mM sodium orthovanadate.
After centrifugation (40 000g, 1 hour, 4°C), the supernatant was applied to a
column of wheat germ agglutinin–Sepharose 4B equlibrated with 130 mM
NaCl, 10 mM Tris-HCl (pH 7.4) (buffer D). The column was washed
thoroughly with buffer D containing 0.2% octanoyl-N-methylglucamide
(ONMG). The bound material was eluted with 2.5% N-acetylglucosamine
in 10 mM Tris, 30 mM NaCl (pH 7.4) (buffer E) containing 0.2% ONMG.
Fractions containing eluted membrane glycoproteins were pooled and
loaded on the echicetin affinity chromatography column equilibrated with
buffer D. The column was washed thoroughly with buffer D containing
0.2% ONMG. The echicetin-Sepharose with bound platelet proteins was
boiled for 1 minute with buffer E containing 1% SDS. Eluted proteins were
separated by electrophoresis and transferred to PVDF membrane.
Protein sequencing
Proteins were separated by SDS-PAGE and blotted to PVDF membrane.
Protein bands were identified by staining parallel lanes, and the corresponding membrane piece was cut out and the protein sequenced on an Applied
Biosystems model 477A pulsed liquid-phase protein sequencer with a
model 120A online phenylthiohydantoin amino acid analyzer.
Flow cytometry
Samples were analyzed using a Becton Dickinson FACScan flow cytometer
(Becton Dickinson, Heidelberg, Germany). Excitation was with an argon
laser at 488 nm. The FACScan was used in a standard configuration with a
530 nm bandpass filter. Standard beads containing specific amounts of
“mean equivalent soluble fluorescein molecules” were used for calibration.
Standard beads or platelets were gated, and data were obtained from
fluorescence channels in a logarithmic mode. A total of 5000 events were
analyzed. Specific binding of antibodies was calculated by substracting
unspecific binding as determined with a FITC-labeled mouse isotypespecific IgG or FITC-labeled chicken IgY. Specific binding of FITC-labeled
fibrinogen was calculated by substracting unspecific binding as determined
with a 10-fold excess of unlabeled fibrinogen.
P-selectin expression and fibrinogen binding to platelets
Washed platelets were diluted to 5 ⫻ 107/mL with HEPES buffer (buffer
C). Platelets (100 ␮L) were activated with echicetin-IgM␬ (5 ␮g/mL
echicetin, 1 ␮g/mL IgM␬) for 5 minutes or thrombin (1 U/mL) in the
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BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
ECHICETIN AGGLUTINATES PLATELETS VIA GPIb AND IgM
2335
presence of GPRP (1.25 mM) for 3 minutes and fixed with formaldehyde.
After platelets were washed and resuspended in 10 mM Tris-HCl, pH 7.4,
buffer, they were incubated with anti-CD62–FITC chicken antibodies (10
␮g/mL). After 1 hour, platelets were again washed and analyzed by
flow cytometry.
In the presence of GPRP (1.25 mM), washed platelets (100 ␮L,
5 ⫻ 107/mL in buffer C) were incubated with fibrinogen-FITC (100
␮g/mL) for 10 minutes. Platelets were activated with echicetin-IgM␬ (5
␮g/mL echicetin, 1 ␮g/mL IgM␬) for 5 minutes or 1 U/mL thrombin for 3
minutes and fixed with formaldehyde. After platelets were washed and
resuspended in Tris buffer, they were analyzed by flow cytometry.
Immunoprecipitation
For immunoprecipitation, aliquots (700 ␮L, 5 ⫻ 108/mL) of control, resting
platelets as well as activated platelets were solubilized in phosphatebuffered saline containing 1.2% Triton X-100 with 1 mM phenylmethylsulfonyl fluoride, 5 mM ethylenediaminetetraacetic acid (EDTA), 2 mM
N-ethylmaleimide, 2 mM benzamidine, and 2 mM sodium orthovanadate.
After centrifugation, platelet lysates precleared with protein A–Sepharose
were stirred for 2 hours with specific antibodies before the addition of 20
␮L protein A–Sepharose followed by 6 to 8 hours of incubation.
Purification of echicetin-binding protein from blood plasma
Human blood plasma was depleted in fibrinogen, dialyzed against 50 mM
Tris-HCl, pH 7.5, and loaded on a Fractogel EMD TMAE-650(S) column
(10 ⫻ 150 mm, Merck) equilibrated with the same buffer. Echicetinbinding protein was eluted by a gradient of NaCl (0-1 mM in Tris buffer).
Fractions (5 mL) were collected at 1 mL/min flow rate. Fractions containing
echicetin-binding protein activity were pooled and purified further by
affinity chromatography on an echicetin–Sepharose 4B column. Echicetinbinding protein was eluted from the echicetin-Sepharose with 100 mM
citrate buffer, pH 2.5.
Figure 2. Inhibition of aggregation of washed platelets induced by vWF or
alboaggregin A. Upper curves: washed human platelets (500 ␮L, 5 ⫻ 108/mL) were
stirred at 1100 rpm at 37°C and aggregation was induced by 5 ␮g/mL human vWF
plus 0.5 mg/mL ristocetin (A) or by 0.1 ␮g/mL alboaggregin A (B). Lower curves:
washed human platelets (500 ␮L, 5 ⫻ 108/mL) were stirred in the presence of 20
␮g/mL echicetin, and the same agonists (A and B) were added after 1 minute.
Results
Echicetin binds specifically to GPIb on the platelet surface
Figure 1. Binding of platelet proteins to echicetin-Sepharose. Platelet surface
proteins were labeled with biotin, platelets were lysed by Triton X-100, and the lysate
was added to echicetin-Sepharose. The proteins eluted from the echicetinSepharose were separated by SDS-PAGE, transferred to PVDF membrane, and
detected with avidin-phosphatase conjugate (lanes 1-3) or with anti-GPIb monoclonal
antibody (lanes 4-6). Lanes 1 and 4: platelet lysates. Lanes 2 and 5: eluate from
Sepharose (negative control). Lanes 3 and 6: eluate from echicetin-Sepharose.
Specific bands detected by anti-GPIb monoclonal antibody (Ib-4) are indicated by
(nonreduced [NR]) GPIb and glycocalicin (GC; the extracellular proteolytic fragment
of GPIb␣) and (reduced [R]) GPIb␣ and macroglycopeptide (MG; the mucinlike
proteolytic fragment of GPIb␣). Under reducing conditions GC and GPIb␣ comigrated.
To establish which platelet receptor binds to echicetin, platelet
surface proteins were labeled with biotin. A fraction enriched in
platelet glycoproteins was prepared by affinity chromatography on
a wheat germ agglutinin–Sepharose 4B column. This fraction was
used for affinity chromatography on echicetin–Sepharose 4B or on
Sepharose 4B as a control. The proteins bound to echicetin or
Sepharose 4B were eluted and separated by gel electrophoresis.
Proteins were transferred to a PVDF membrane, and the membrane
was treated with anti-GPIb mAb (Ib-4), peroxidase-coupled goat
antimouse second antibodies, and bound antibodies were detected
by chemiluminescence. The membrane was restained with avidinphosphatase conjugate to identify biotinylated platelet membrane
proteins, which were bound to echicetin or Sepharose 4B. The
results of this experiment are shown in Figure 1. Echicetin–
Sepharose 4B bound only GPIb and some of its proteolytic
degradation products among the platelet membrane proteins.
Sepharose 4B alone did not bind any membrane proteins from
platelet lysate.
Echicetin-induced agglutination of platelets in plasma
High-purity echicetin isolated from Echis carinatus venom was
tested for its ability to inhibit platelet aggregation induced by vWf
and alboaggregin A as well as by low doses of thrombin. This
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2336
NAVDAEV et al
Figure 3. Echicetin-induced platelet agglutination in blood plasma independent
of GPIIb/IIIa. PRP (500 ␮L, curve 1) or washed human platelets (500 ␮L,
5 ⫻ 108/mL, curve 3) were stirred at 1100 rpm at 37°C, and echicetin (5 ␮g) was
added to each sample. Curve 2: platelet agglutination in PRP (500 ␮L) induced by
echicetin (5 ␮g) in the presence of GPIIb/IIIa inhibitor (Ro44-9883, 1 ␮M/mL).
echicetin preparation had the same properties as those previously
described.9 Echicetin (20 ␮g/mL) completely inhibited aggregation
of washed platelets induced by vWf (5 ␮g/mL) plus ristocetin (0.5
mg/mL) or by alboaggregin A (0.1 ␮g/mL) (Figure 2 A,B).
It was previously reported9 that intravenous injection of echicetin in small animals to test for antihemostatic or antithrombotic
effects can provoke thrombocytopenia. Therefore, we investigated
the action of echicetin on PRP. In contrast to experiments with
washed platelets, where no agglutination was seen, in blood plasma
echicetin induced platelet agglutination (Figure 3). Whether echicetin and an echicetin-binding protein from plasma simply agglutinate platelets or whether, in addition, aggregation occurs by
activation of IIb/IIIa receptors and formation of fibrinogen bridges
between platelets was not clear. Therefore, a IIb/IIIa inhibitor was
used to prevent fibrinogen binding to platelets, but it did not affect
platelet agglutination induced by echicetin in plasma (Figure 3).
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
cytometry. After activation, platelets were fixed with formaldehyde, washed with Tris buffer, and stained by FITC-labeled
anti–P-selectin antibodies (10 ␮g/mL). The amount of antibodies
bound was measured by flow cytometry. Binding of anti–P-selectin
antibodies increased strongly on both thrombin and echicetin-IgM␬–
activated platelets. The thrombin-activated platelets expressed
higher levels of P-selectin than those activated with echicetinIgM␬ (Figure 4A).
To investigate GPIIb/IIIa activation, FITC-labeled fibrinogen
(100 ␮g/mL) was added to a suspension of 100 ␮L of washed
platelets in the presence of GPRP (1.25 mM), and platelets were
activated as described above. After activation, platelets were fixed
with formaldehyde and washed with Tris buffer, and the amount of
bound FITC-fibrinogen was measured by flow cytometry.
Binding of fibrinogen-FITC increased on the surface of echicetinIgM␬– or thrombin-activated platelets compared with resting
platelets. Again, fibrinogen binding increased more strongly
on platelets activated with thrombin than with echicetin-IgM␬
(Figure 4B).
The increased fluorescence found with fibrinogen and anti–Pselectin antibodies after activation could possibly have been an
artifact due to platelet agglutination by echicetin-IgM␬ rather than
a real increase in P-selectin expression and GPIIb/IIIa activation.
Therefore, as a control, binding of anti-CD36 antibodies (10
␮g/mL) to platelets activated under the same conditions was
examined. It was shown previously that levels of CD36 do not
change appreciably on the surface of activated platelets compared
Echicetin binds specifically to plasma IgM with ␬ light chain
To identify the plasma component that binds to echicetin, plasma
was fractionated by ion-exchange chromatography on a TMAEFractogel column followed by affinity chromatography of the
active fractions on an echicetin–Sepharose 4B column. Eluates
from this column contained a protein that showed a high molecular
mass single band on SDS-PAGE under nonreduced conditions and
2 bands with masses of 70 kd and 25 kd under reduced conditions.
The N-terminal amino acid sequence of the 70 kd chain was
EVQLVESGGXL, which is typical for the variable III domain of
the heavy chain of immunoglobulins. This protein was analyzed
further by dot blot using specific antiheavy and antilight chain
immunoglobulin antibodies. Antibodies to ␮ heavy chain and ␬
light chain bound specifically to this protein. Thus, the protein
isolated from plasma that specifically binds echicetin is IgM with a
␬ light chain.
P-selectin expression and fibrinogen binding to platelets
activated by echicetin-IgM␬ complex
The expression of P-selectin on platelets after 5 minutes of
activation by echicetin-IgM␬ (5 ␮g/mL echicetin, 1 ␮g/mL IgM␬)
or thrombin (1 U/mL) (as positive control) was determined by flow
Figure 4. P-selectin expression and fibrinogen binding to platelets activated by
echicetin-IgM␬. Platelets were activated by echicetin-IgM␬ or thrombin as positive
control, and binding of anti–P-selectin antibodies (A) or FITC-fibrinogen (B) was
measured. In comparison to resting platelets (1), both echicetin-IgM␬–activated (2)
and thrombin-activated (3) platelets bind higher amounts of anti P-selectin antibodies
and FITC-fibrinogen. All measurements were repeated with platelets from 3 different donors.
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BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
ECHICETIN AGGLUTINATES PLATELETS VIA GPIb AND IgM
2337
of IIb/IIIa receptor (Ro44-9883, 1 ␮M/mL) was added to the
platelet suspension 1 minute before adding echicetin-IgM␬. There
was no difference in agglutination response between inhibited
platelets and untreated platelets. The IIb/IIIa inhibitor also had no
effect on protein tyrosine phosphorylation in platelets activated by
echicetin-IgM␬ (data not shown).
EDTA (5 mmol/mL) did not affect platelet agglutination
induced by echicetin-IgM␬; however, EDTA slightly suppressed
tyrosine phosphorylation of the 70- to 90-kd proteins (Figure 7).
Figure 5. Effect of GPIIb/IIIa inhibitor and ADP receptor inhibitor on fibrinogen
binding to platelets. Platelets pretreated with GPIIb/IIIa inhibitor (Ro44-9883, 1
␮M/mL) or ADP receptor inhibitor (AR-C66096, 1 ␮M/mL) were activated with
echicetin-IgM␬ or thrombin as positive control, and binding of FITC-labeled fibrinogen
was examined compared with resting platelets. All measurements were repeated 3
times with platelets from different donors. Graph shows fibrinogen binding to resting
platelets (1), echicetin-IgM␬–activated platelets without inhibitors (2), echicetin-IgM␬–
activated platelets with IIb/IIIa inhibitor (3), echicetin-IgM␬–activated platelets with
ADP receptor inhibitor (4), thrombin-activated platelets without inhibitors (5), thrombinactivated platelets with IIb/IIIa inhibitor (6), and thrombin-activated platelets with ADP
receptor inhibitor (7).
with resting platelets.20 We also did not find any marked differences
in expression level of CD36 on activated platelets in our experiments (data not shown).
The specific GPIIb/IIIa inhibitor Ro44-9883 21 at 1 ␮mol/mL
and ADP receptor inhibitor AR-C66096 22 at 1 ␮mol/mL were used
to investigate the role of fibrinogen binding to platelets as well as
involvement of ADP in GPIIb/IIIa activation in platelets stimulated
by echicetin-IgM␬ complex. Thrombin-activated platelets were
used as a positive control. Ro44-9883 was able to completely
inhibit fibrinogen binding to both echicetin-IgM␬– and thrombinactivated platelets (Figure 5). ADP receptor inhibitor slightly
decreased binding of fibrinogen to the surface of echicetin-IgM␬–
activated platelets but had no effect on binding of fibrinogen to
thrombin-activated platelets (Figure 5).
Protein tyrosine phosphorylation in platelets activated by
echicetin-IgM␬ complex
Echicetin alone at concentrations up to 20 ␮g/mL did not induce
the agglutination of washed platelets. However, addition of echicetin-binding IgM␬ to platelet suspensions containing echicetin
induced agglutination (Figure 6A). Aliquots of platelets at various
times after addition of IgM␬ were lysed by SDS and examined for
protein tyrosine phosphorylation. Echicetin-IgM␬ complex induced marked changes in tyrosine phosphorylation of several
platelet proteins with masses of 64, 70 to 90, and 120 kd (Figure
6B). The tyrosine phosphorylation of these proteins increased
rapidly after addition of IgM␬ but was not affected by echicetin
alone. Fc␥ and p44, which show strongly increased tyrosine
phosphorylation in platelets in response to alboaggregin A,23 were
not tyrosine phosphorylated in response to echicetin-IgM␬ (Figure 6B).
Influence of EDTA, IIb/IIIa inhibitor, and acetylsalicylic acid on
activation of platelets by echicetin-IgM␬ complex
To study the involvement of fibrinogen binding in the aggregation
of washed platelets by echicetin-IgM␬ complex, a specific inhibitor
Figure 6. Agglutination and protein tyrosine phosphorylation induced by
echicetin-IgM␬ complex in washed human platelets. (A) Washed human platelets
(500 ␮L, 5 ⫻ 108/mL) were stirred at 1100 rpm at 37°C in the presence of 5 ␮g
echicetin. One minute after adding echicetin, 1 ␮g IgM␬ (upper curve) or buffer for
control (bottom curve) were added. (B) Proteins from SDS-lysed platelets were
separated by SDS-PAGE, transferred to PVDF membrane, and stained with antiphosphotyrosine antibody (4G10). The left panel shows proteins from echicetin-treated
platelets; the right panel shows tyrosine phosphorylation of proteins from platelets
activated by echicetin-IgM␬ complex.
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2338
NAVDAEV et al
Figure 7. Influence of EDTA on platelet agglutination and activation by
echicetin-IgM␬ complex. (A) Washed human platelets (500 ␮L, 5 ⫻ 108/mL) were
stirred at 1100 rpm at 37°C in the presence (upper line) or absence (bottom line) of 5
mM EDTA and agglutinated by echicetin (5 ␮g) plus IgM␬ (1 ␮g). (B) After 2 minutes
of agglutination with echicetin-IgM␬ complex, platelets were lysed by SDS. Proteins
were separated by SDS-PAGE, transferred to a PVDF membrane, and stained with
antiphosphotyrosine antibody (4G10).
Platelets incubated with acetylsalicylic acid (100 mM/mL) for 5
minutes before adding echicetin-IgM␬ did not show differences in
platelet agglutination or protein tyrosine phosphorylation compared with control platelets (data not shown).
Tyrosine kinases p72SYK and p53/56LYN but not p125FAK are
involved in platelet activation by echicetin-IgM␬
Because agglutination of platelets by echicetin-IgM␬ complex
induced clear changes in tyrosine phosphorylation of several
proteins, the involvement of candidate tyrosine kinases p72SYK,
p53/56LYN, and PI-3K were investigated. Washed platelets were
activated by echicetin-IgM␬ (5 ␮g/mL echicetin, 1 ␮g/mL IgM␬),
lysed in Triton X-100 (1.2%), and centrifuged to remove the
cytoskeleton. Specific antibodies against p72SYK, p53/56LYN, and
PI-3K with protein A–Sepharose were used for immunoprecipitation from the supernatant of platelet lysates.
Activation of all of these kinases has been shown to be
associated with tyrosine phosphorylation. Tyrosine phosphorylation of p72SYK and p53/56LYN increased rapidly after activation of
platelets by echicetin-IgM␬ (Figure 8A). Tyrosine phosphorylation
of p72SYK markedly increased for 30 seconds after adding IgM␬
and then continued to increase slowly.
In contrast to p72SYK, phosphorylation of p53/56LYN increased
rapidly for the first 30 seconds, a maximum, and then rapidly
decreased. At the same time, the amount of p53/56LYN in the
supernatant of platelet lysates also decreased. This decrease was
due to p53/56LYN binding to cytoskeletal proteins (Figure 8B) and
therefore probably not due to dephosphorylation.
There were no changes in tyrosine phosphorylation of PI-3K in
response to echicetin-IgM␬. Platelets treated with wortmannin (1
␮M), a specific inhibitor of PI-3K, for 5 minutes also did not show
any differences in response to echicetin-IgM␬. These data support a
role for p72SYK and p53/56LYN but not PI-3K in activation of
platelets by the echicetin-IgM␬ complex.
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
Figure 8. Tyrosine phosphorylation of p72SYK and p53/56LYN in platelets
activated by echicetin-IgM␬ complex. After activation, platelets were lysed by
1.2% Triton X-100 and cytoskleton was removed by centrifugation at 100 000g.
Supernatant was used for immunoprecipitation by specific anti-p72SYK (A) or
anti-p53/56LYN (B) antibodies coupled to protein A–Sepharose 4B. Immunoprecipitated proteins were eluted by 1% SDS, separated by SDS-PAGE, transferred to
PVDF membrane, and stained with antiphosphotyrosine antibody (4G10) or with
specific anti-p72SYK and anti-p53/56LYN antibodies. Proteins from cytoskeleton were
solubilized in 1% SDS, separated by SDS-PAGE, transferred to PVDF membrane,
and stained with anti-p53/56LYN antibodies.
Activation and tyrosine phosphorylation of p125FAK as a result of
signaling through activated and clustered GPIIb/IIIa was shown earlier.24 We examined tyrosine phosphorylation of p125FAK in platelets
activated by echicetin-IgM␬ to study any involvement of GPIIb/IIIa
signaling. Activated, washed platelets were lysed with Triton X-100,
p125FAK was immunoprecipitated, and tyrosine phosphorylation determined using 4G10 antibody. No changes in tyrosine phosphorylation of
p125FAK in echicetin-IgM␬–activated platelets were detected compared
to resting platelets (data not shown).
Figure 9. Inhibition of platelet agglutination induced by echicetin-IgM␬. Washed
human platelets (500 ␮L, 5 ⫻ 108/mL) were stirred at 1100 rpm at 37°C. A total of 5
␮g echicetin was added to the platelet suspension and incubated for 1 minute.
Agglutination was started by adding 1 ␮g IgM␬. Monoclonal antibody against GPIb
was added to platelets 2 minutes before echicetin. Curve 1: platelet agglutination
induced by echicetin-IgM␬ without any inhibitors. Curve 2: in the presence of 16
␮g/mL SZ2. Curve 3: in the presence of 3.8 ␮g/mL VM16d. Curve 4: in the presence
of 11.4 ␮g/mL VM16d.
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BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
Echicetin-IgM␬ complex agglutinates and activates platelets
through GPIb only
Activation of washed platelets by echicetin-IgM␬ complex is
probably the result of GPIb clustering. However, immunoglobulins
complexed with echicetin could possibly activate other platelet
receptors. To confirm the essential role of GPIb in this process,
monoclonal antibodies SZ2 and VM16d, which bind to different
sites on GPIb molecule, were used to inhibit platelet agglutination
induced by echicetin-IgM␬. SZ2 inhibited platelet agglutination
only slightly even at high concentrations (Figure 9, curve 2).
However, VM16d completely inhibited the agglutination. The
inhibition was dependent on the VM16d mAb concentration in the
sample (Figure 9, curves 3 and 4).
An alternative approach to clustering GPIb using biotinylated
echicetin cross-linked by avidin was investigated. Biotin was
coupled to echicetin to give a biotin:echicetin molar ratio of 1.5:1.
The ability of biotinylated echicetin to bind to the surface of fixed,
washed platelets was examined by flow cytometry. Biotinylated
echicetin binds to the surface of fixed platelets in a saturable
manner. Binding of biotinylated echicetin to fixed platelets was
inhibited by an excess of unlabeled echicetin (data not shown). It
was shown previously that alboaggregin A can agglutinate fixed
platelets by binding to GPIb.4 We found that 0.2 ␮g/mL alboaggregin A agglutinated fixed platelets to give visible aggregates.
Echicetin added to a suspension of fixed platelets 5 minutes before
alboaggregin A inhibits this agglutination in a dose-dependent
manner. Echicetin at a concentration of 20 ␮g/mL completely
blocks the agglutination of fixed platelets by alboaggregin A (0.2
␮g/mL). There were no differences in ability to inhibit alboaggregin A–dependent agglutination of fixed platelets between biotinylated echicetin and unlabeled echicetin (data not shown).
Biotinylated echicetin was used in a similar way to echicetinIgM␬ to activate platelets by cross-linking with avidin. The
biotinylated echicetin/avidin complex induced agglutination of
washed human platelets (Figure 10, curve 1) as effectively as
echicetin-IgM␬ or echicetin in PRP (compare with Figures 3 and
6A). Neither GPIIb/IIIa inhibitor nor EDTA blocked platelet
agglutination induced by biotinylated echicetin and avidin (Figure
10, curves 2 and 3).
Figure 10. Agglutination of platelets induced by biotinylated echicetin/avidin
complex. Washed human platelets (500 ␮L, 5 ⫻ 108/mL) were stirred at 1100 rpm at
37°C. Biotinylated echicetin (5 ␮g) was added to the platelet suspension and
incubated for 1 minute. Agglutination was started by adding 2 ␮g avidin. Curve 1:
platelet agglutination induced by biotinylated echicetin/avidin. Curve 2: GPIIb/IIIa
inhibitor (Ro44-9883, 1 ␮M/mL) was added to platelet suspension 1 minute before
adding of biotinylated echicetin/avidin. Curve 3: EDTA (5 mM/mL) was added to
platelet suspension 5 minutes before adding biotinylated echicetin/avidin. Curve 4:
washed platelets plus biotinylated echicetin without avidin.
ECHICETIN AGGLUTINATES PLATELETS VIA GPIb AND IgM
2339
Protein tyrosine phosphorylation in platelets activated by biotinylated echicetin/avidin complex was also similar to that obtained
with platelet activation by echicetin-IgM␬ (data not shown).
Discussion
A number of proteins from different snake venoms bind to platelet
GPlb. Some of these, such as flavocetin A and mamushigin, have
been shown to activate platelets. Echicetin itself does not activate
washed platelets but inhibited platelet activation by vWF, thrombin, or alboaggregin A9,14 (Figure 2). It is also known that echicetin
can induce thrombocytopenia after injection into mice.9,15 This
observation was previously unexplained. We found that platelets in
PRP, unlike washed platelets, agglutinate in the presence of
echicetin (Figure 3). Plasma was therefore fractionated by ion
exchange chromatography and the fractions identified that induce
agglutination of washed platelets in the presence of echicetin. A
final purification to a single band (nonreduced) on SDS-PAGE was
obtained by affinity chromatography on an echicetin–Sepharose 4B
column. The purified product had a very high molecular mass
nonreduced (⬎ 500 kd) and reduced gave 2 bands at 70 and 25 kd.
The N-terminal amino acid sequence for the 70-kd chain was found
to be EVQLVESGGXL, which is typical for the variable III domain
of the heavy chain of IgG and IgM. These results suggested that the
protein was an immunoglobulin, and it was thus tested against a
panel of heavy and light chain immunoglobulin specific antibodies.
Antibodies to ␮ heavy chains and ␬ light chains gave a clear
positive response whereas others were negative, indicating that the
purified plasma protein binding to echicetin was IgM with ␬ light
chains. This also explains the clustering of echicetin in plasma.
Because IgM is pentameric, theoretically up to 5 molecules of
echicetin can bind to one molecule of IgM. This mechanism can
cluster several molecules of echicetin attached to the surface of one
platelet and, consequently, cluster GPIb receptors. On the other
hand, it can bind molecules of echicetin on the surface of different
platelets and provide a mechanism for the agglutination of platelets
(Figure 11A). This mechanism can explain the thrombocytopenia
observed in mice after echicetin injection. In this case the increase
in the bleeding time can be influenced by the decrease in platelet
count as well as by inhibition of vWf/thrombin platelet activation
by echicetin.
Binding of echicetin-IgM␬ complex to platelets induced agglutination and partial activation of washed platelets. However, neither
EDTA nor GPIIb/IIIa inhibitor prevent platelet agglutination in
response to echicetin-IgM␬, raising the question whether GPIIb/
IIIa is activated. It was shown before that GPIIb/IIIa can be
activated and can bind fibrinogen without aggregation necessarily
occurring.16,25 FITC-fibrinogen binding to platelets activated by
echicetin-IgM␬ was examined by flow cytometric analysis and was
significantly increased on activated platelets (Figure 4).
GPIIb/IIIa inhibitor completely abolished binding of FITCfibrinogen to the surface of platelets activated by echicetin-IgM␬
(Figure 5). These data show that GPIIb/IIIa is activated on the
surface of these stimulated platelets. However, neither GPIIb/IIIa
inhibitor nor EDTA affect agglutination/aggregation of platelets by
either echicetin-IgM␬ (Figure 3) or biotinylated echicetin/avidin
(Figure 10), demonstrating that aggregation via GPIIb/IIIafibrinogen does not occur. The mechanism of GPIIb/IIIa activation
via GPIb is still far from clear. One possibility is direct activation of
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2340
NAVDAEV et al
Figure 11. Mechanism of platelet agglutination and activation induced by
echicetin-IgM complex or biotinylated echicetin/avidin. Echicetin binds to GPIb.
(A) One molecule of IgM␬ can bind up to 5 molecules of echicetin. Binding of several
molecules to the surface of one platelet results in clustering of GPIb molecules (1).
Binding of echicetin molecules attached to the surface of different platelets results in
agglutination (2). (B) One molecule of avidin can bind up to 4 molecules of biotin.
Binding of several molecules of biotinylated echicetin to the surface of one platelet
results in clustering of GPIb molecules (1). Binding of biotinylated echicetin molecules attached to the surface of different platelets results in agglutination (2).
GPIIb/IIIa after GPIb clustering.26 Alternatively, GPIIb/IIIa activation may largely result from ADP receptor activation by ADP
released from granules. Activation of platelets by echicetin-IgM␬
also induces granule release as assessed by P-selectin expression
measured by flow cytometry. Thus, ADP is released as well. We
have examined the possibility that ADP is involved in GPIIb/IIIa
activation by inhibition of the P2T ADP receptor. Some decrease
(about 20%) in FITC-fibrinogen binding to the surface of platelets
was observed. However, ADP receptor inhibition did not completely inhibit FITC-fibrinogen binding to platelets activated by
echicetin-IgM␬. ADP receptor inhibitor did not prevent FITC-
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
fibrinogen binding to platelets activated by thrombin. These data
suggest that probably both mechanisms, direct activation of
GPIIb/IIIa via clustering of GPIb as well as activation of GPIIb/IIIa
by feedback through ADP, operate in platelets activated by
echicetin-IgM␬. Clustering of GPIb by vWf has been previously
proposed as a mechanism for initial activation of platelets under
high shear stress conditions. Several articles support this hypothesis, including that of Falati et al23 where alboaggregin A and
mutant forms of vWf were used to cluster GPIb. The authors
showed that alboaggregin A induced tyrosine phosphorylation of
Syk, Fyn, Lyn, phospholipase C␥2, Fc␥, and proteins with mass 44,
56, and 59 kd. Platelet activation by echicetin-IgM␬ (or biotinylated echicetin/avidin) also caused tyrosine phosphorylation of Syk
and Lyn. However, in the experiments with platelets activated via
echicetin clustering we did not find tyrosine phosphorylation of
Fc␥ and p44. These differences in experimental results may be due
to binding of alboaggregin A to more than one class of receptor on
the platelet surface. Strong activation of Fc␥ in platelet activation
by alboaggregin A23 might be induced via binding to GPVI, for
example. Platelet activation by clustering GPIb receptors was also
examined by Yanabu et al.27 In this case receptors were clustered
using a GPIb-specific antibody NNKY5-5. The authors reported
that activation of platelets in blood plasma by NNKY5-5 caused
formation of small aggregates and tyrosine phosphorylation of
p72SYK and a protein with mass of 64 kd. However, washed
platelets only showed a minimal response to NNKY5-5.
In our experiments, cross-linking–washed platelets by echicetinIgM␬ or PRP by echicetin or by biotinylated echicetin/avidin
induced very similar changes in light transmission, implying that
the size of the aggregates in PRP and with washed platelets was
similar as well. Visual inspection of the aggregates supported this
interpretation. This implies a similar mechanism in each case,
without participation of other plasma components, limited by some
common factor such as GPIb density on platelets.
It was also shown that inhibition of GPIIb/IIIa by GRGDS
peptide or by a specific monoclonal antibody completely suppressed platelet aggregation induced by NNKY5-5. This shows that
GPIIb/IIIa was involved in aggregation, indicating that platelet
activation had occurred. It was shown earlier that fibrinogen
binding to activated GPIIb/IIIa induced the activation of p72SYK.28,29
In contrast to the data of Yanabu et al,27 we did not find any
involvement of GPIIb/IIIa clustering by fibrinogen in the process
of platelet agglutination/activation induced by echicetin-IgM␬ (or
biotinylated echicetin/avidin complex). However, we also found
tyrosine phosphorylation of p72SYK and p64. It has also been shown
that vWf can induce tyrosine phosphorylation of p72SYK and p64 in
platelets independently of GPIIb/IIIa.30 Thus, the mechanism of
tyrosine phosphorylation of p72SYK and p64 induced by NNKY5-5
is unclear, because the phosphorylation could be a result of
signaling from either GPIb or GPIIb/IIIa or both.
Recently, Zaffran et al26 and Yap et al31 have shown that GPIb
complexes transfected into Chinese hamster ovary cells, which
already have GPIIb/IIIa transfected, are able to transmit signals to
activate GPIIb/IIIa. The mechanisms involved are not clear and
seem to depend upon the shear stress involved, lower shear being
compensated by release and feedback of ADP and thromboxanes.
In general, our results support these conclusions and suggest that
signal transduction by engagement of GPIb alone in platelets is
capable of activating GPIIb/IIIa to bind fibrinogen. Aggregation
and further signaling via GPIIb/IIIa may require the cross-linking
of GPIb with GPIIb/IIIa that normally occurs with vWf.
In conclusion, we have shown that echicetin can bind IgM␬
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BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
ECHICETIN AGGLUTINATES PLATELETS VIA GPIb AND IgM
from blood plasma. The complex of echicetin-IgM␬ effectively
induces platelet agglutination, which is not dependent on fibrinogen binding to GPIIb/IIIa. Cross-linking of GPIb by the echicetinIgM␬ complex also induces tyrosine phosphorylation of p72SYK,
p53/56LYN, p64, p70 to p90, and p120. The echicetin-IgM␬
complex should be a good reagent for exploring signal transduction
mechanisms induced via GPIb complex independently of other
platelet receptors. These results also suggest that the mechanisms
of action of other inhibitory snake C-type lectins that bind to GPIb
may require reinvestigation.
2341
Acknowledgments
We thank Dr Edith Magnenat, Serono Pharmaceutical Research
Institute, Geneva, Switzerland, for the peptide sequencing; Prof
Beda Stadler, Department of Clinical Research, University of
Berne, Switzerland, for the immunoglobulin analyses; and the
Central Laboratory of the Swiss Red Cross Blood Transfusion
Service for the supply of buffy coats, erythrocyte concentrates, and
IgM fractions.
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2001 97: 2333-2341
doi:10.1182/blood.V97.8.2333
Echicetin, a GPIb-binding snake C-type lectin from Echis carinatus, also
contains a binding site for IgM κ responsible for platelet agglutination in
plasma and inducing signal transduction
Alexei Navdaev, Dagmar Dörmann, Jeannine M. Clemetson and Kenneth J. Clemetson
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